Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

Background The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. Results The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. Conclusions A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12936-024-04862-w.

2. Coat dishes containing gridded coverslips (MatTek, P35G-1.5-14-CGRD) with poly-D-lysine (PDL): a. Add 2mL of 50 μg/mL of poly-D-lysine (Gibco) diluted in Dulbecco's Phosphate Buffered Saline (D-PBS; Gibco) in each dish and incubate for 1h at room temperature (RT) b.Wash 3 times with 2 mL of D-PBS c. Remove any traces of liquid and dry, with lids off, in a biosafety cabinet for 2h d.Use immediately or store at 4 o C 3. Detach and seed cells (from step 1) using Trypsin-EDTA (0.25%; Gibco) at 700,000 cells per PDL-coated dish in 2.5 mL of Huh7 medium, one day prior to the infection.
4. Using microdissection, isolate sporozoites from the salivary glands of Anopheles mosquitoes infected with P. berghei, as previously described with modifications [1,2].Mosquitoes infected with P. berghei ANKA GFP-LUCCON [3] were produced by the SporoCore (University of Georgia) and used for this study.
6. Spin-infect Huh7 cells with 2mL of sporozoite suspension at 330 × g for 3 min, using low brake and acceleration settings.
7. Incubate cells for 2h at 37 o C and 5% CO2.Change the cell culture medium with fresh Huh7 medium and further incubate infected cells at 37 o C and 5% CO2 until the desired time point.4. Dilute sporozoite to desired concentration (e.g., 0.5-1 million sporozoites per dish) in 1% PSN Hep medium.

Infection of primary hepatocytes with
5. Spin-infect hepatocytes with 2 mL of sporozoite suspension at 200 × g for 5 min, using low brake and acceleration settings.
6. Incubate cells for 2h at 37 o C and 5% CO2.Change cell culture medium with 2.5 mL of fresh 1% PSN Hep medium and further incubate infected cells at 37 o C and 5% CO2.
7. Next day and every other day, change medium with 5% PSN Hep medium and further incubate infected cells at 37 o C and 5% CO2 until the desired time point.
2. Wash cells once with Imaging medium.
3. Generate fiducial markers by scraping off linear patterns of cells from the monolayers attached to the gridded coverslips.This step is required since hepatocyte monolayers are too thick to allow the visualization of the gridded alpha-numerical labeling of the coverslips.Gentle scraping was conducted with a thin needle.6. Wash cells twice with D-PBS.
7. Generate fiducial markers by scraping off linear patterns of cells from the monolayers attached to the gridded coverslips.This step is required since hepatocyte monolayers are too thick to allow the visualization of the gridded alpha-numerical labeling of the coverslips.Gentle scraping was conducted with a thin needle.Wash cells twice with D-PBS.
8. Perform imaging at RT using light and fluorescence microscopy.For each ROI, acquire images at a range of magnifications (e.g., using 4×, 10×, 20× and 40× objectives) to facilitate the tracking of ROIs and the correlation between light and electron microscopy images.9. Wash samples twice with D-PBS and incubate samples for 15min at RT in D-PBS containing 2% paraformaldehyde and 2% glutaraldehyde (EMS).
10. Keep samples at 4 o C, in the dark and in the fixative solution, until processing for CLEM.

Preparation of samples for TEM imaging
1. Rinse samples 3 times for 5 min each at RT in PBS, pH 7.4.During all steps, place the small MatTek plates inside a larger petri dish to reduce direct handling.Using a scribe pen, mark the side wall of each plate with identifying information.
2. Immerse samples in 1% osmium tetroxide and 1.6% potassium ferricyanide in PBS for 30 min.Keep covered from light, no agitation required.
3. Rinse samples in PBS 3 times and then once in distilled water for 5 min each at RT.
5. Progressively infiltrate samples, while rocking, with normal hardness Epon resin (EMS) with BDMA accelerant.Infiltrate with 100% resin 3 times for 30-60 min each.Ensure that only a thin amount of resin remains within the glass bottom dishes to enable the best possible chance for separation of the glass coverslip.To do this, slowly pour off all resin that is possible to remove (do not pipette as this may introduce air bubbles).Add back fresh resin one drop at a time to the inner well of the MatTek dish until the surface of the well is just slightly filled below the layer of surrounding plastic.Place the dishes onto cardboard and then polymerize at 60 o C for 12-18 hours.
6. Remove glass coverslips using ultra-thin Personna razor blades (EMS) and liquid nitrogen (LN2) exposure, as needed.To remove the coverslip, break the ultra-thin double razor into single blades by pinching the blade in half with the paper cover still on.While holding the MatTek dish upside down (i.e., coverslip side up), slide the single blade between one of the square edges of the coverslip and the plastic.The blade will need to be held with a very slight bend to both assist in applying gentle upward pressure and to ease the blade closer to the surface of the plastic dish (due to the ridge along the bottom of the MatTek plate).With small rocking motions, move the edge of the razor blade across the plastic plate and toward the opposite corner of the coverslip.As this happens, a small border of separation of the coverslip from the resin/plastic will be observed.Propagate that edge slowly but without halting.If the glass breaks, take care in removing the loose glass.Hold the dish coverslip side down and grab the edge of the plate with forceps while lowering the bottom of the dish (and the entire coverslipped region) into the LN2 so that just the bottom of the plate is submerged.Avoid plunging the whole dish into LN2.Once the crackling and popping noises cease, bring the plate back into position to proceed with the ultra-thin razor blade separation method described above.Repeat the LN2 exposure and razor blade separation as needed until the entire coverslip has been removed.
7. Identify ROIs using the gridded alpha-numerical labeling and then mark the ROIs using a diamond or tungsten carbide scribe pen by drawing small boxes around the ROI on the surface of the resin.It is easiest to do this with the aid of a dissection microscope and while the resin it still contained within the MatTek plate.Once the ROI have been marked, the exposed resin surface can be pushed out of the center of the MatTek dish by covering it with a tissue and pushing with a thumb.Take care not to scratch the surface of the resin as that can damage the cells beneath the resin surface.
8. Separate the marked ROIs with ultra-thin razor blades and with the disk of resin held on a glass slide covered with double-sided tape to keep the pieces of resin stable during cutting.
To prepare blocks for sectioning, mount the individual ROIs onto labeled blank resin blocks using cyanoacrylate glue.9. Cut serial thin sections (80-90 nm) using an ultramicrotome from the surface of the block (corresponding to the bottom of the cell layer) until approximately 20 microns in depth to ensure complete capture of the cell volumes.Sequentially collect section-ribbons onto formvar-coated slot grids.During the serial sectioning process, collect a few thicker sections (250-350 nm) onto glass slides.Stain these thicker sections with toluidine blue (T-blue) to track the cells of the ROIs and to serve as a bridge from the fluorescence to the TEM data.
During TEM, it is useful to have images taken of the T-blue stained thick sections and the fluorescence datasets for each ROI.The orientation of the sections on the TEM may have been flipped and/or rotated from the T-blue orientation.Identifying the schizont or hypnozoite structures within a specific cell requires a general mapping and correlation of the surrounding host cell nuclei, a process most easily done with the fluorescence, brightfield, and T-blue images.From that correlation, specific structures of interest can be quickly found via TEM.During TEM imaging, it is helpful to take a series of data covering from very low magnification (20-40 ×) to higher magnification (13,000 ×) to maintain the context from which the higher magnification images, particularly of hypnozoites, are collected.Lastly, when utilizing the serial sections for 3-dimensional context, keep track of the section order during TEM imaging.
12. Correlating images from CLEM datasets can be done with multiple approaches including freehand overlays with Adobe Photoshop or Microsoft PowerPoint.A more guided approach to overlaying can be followed as described by Keene et al. [5].
4. Change medium for fresh Imaging medium and perform imaging at RT using light and fluorescence microscopy.Limit imaging time or use a microscope with an environmental chamber.For each region of interest (ROI), acquire images at a range of magnifications (e.g., using 4×, 10×, 20× and 40× objectives) to facilitate the tracking of ROIs and the correlation between light and electron microscopy images.Incubate samples in D-PBS containing 4% paraformaldehyde for 15 min at RT and wash twice in D-PBS.Store samples at 4 o C in D-PBS, if needed.Incubate samples with primary antibodies for 3h in 2% BSA D-PBS.Primary antibodies used for this study were αUIS4Pb goat IgG (SICGEN; 1:250) or αUIS4Pc human IgG (1:2,000).The αUIS4Pc human IgG was produced in-house and is a mouse-derived antibody inserted into the human antibody backbone [2]. 5. Wash samples 4 times in D-PBS and incubate with secondary antibodies for 90 min in 2% BSA D-PBS containing 2 μg/mL Hoechst at RT. Secondary antibodies used in this study were Alexa Fluor 568 donkey anti-goat IgG or Alexa Fluor 488 goat anti-human IgG (Invitrogen; 5. Wash samples twice with D-PBS and incubate samples for 15 min at RT in D-PBS containing 2% paraformaldehyde (EMS) and 2% glutaraldehyde (EMS).6.Keep samples at 4 o C, in the dark and in the fixative solution, until processing for CLEM.Immunostaining and light and fluorescent microscopy imaging for IFA-CLEM1.1:1,000).